Modular Use of the Uniquely Small Ring A of Mersacidin Generates the Smallest Ribosomally Produced Lanthipeptide

Mersacidin is an antimicrobial class II lanthipeptide. Lanthipeptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs), characterized by intramolecular lanthionine rings. These rings give lanthipeptides their bioactive structure and stability. RiPPs are produced from a gene cluster that encodes a precursor peptide and its dedicated unique modification enzymes. The field of RiPP engineering aims to recombine modification enzymes from different RiPPs to modify new substrates, resulting in new-to-nature molecules with novel or improved functionality. The enzyme MrsM from the mersacidin gene cluster installs the four lanthionine rings of mersacidin, including the uniquely small ring A. By applying MrsM in RiPP engineering, this ring could be installed in linear peptides to achieve stabilization by a very small lanthionine or to create small lanthionine-stabilized modules for chemical modification. However, the formation of unique intramolecular structures like that of mersacidin’s ring A can be very stringent. Here, the formation of ring A of mersacidin is characterized by mutagenesis. A range of truncated mersacidin variants was made to identify the smallest possible construct in which this ring could still be formed. Additionally, mutants were created to study the flexibility of ring A formation. It was found that although the formation of ring A is stringent, it can be formed in a core peptide as small as five amino acids. The truncated mersacidin core peptide CTFAL is the smallest ribosomally produced lanthipeptide reported to date, and it has exciting prospects as a new module for application in RiPP engineering.


S2. List of primers used in this study
In peak 1, TCEP + IAA does not shift the expected mass for the dehydrated construct (S7), meaning that the ring was formed in this peak. In peak 2 most product shifted. In peak 3 the product had the wrong mass and was not further analyzed. In peak 1, the main product does not have the expected mass, probably through an unkown adduct. In peak 2, TCEP + IAA shifts the expected mass for the dehydrated construct (S7), meaning that the ring was mostly not formed in this peak. For this construct atmost a bit of product is formed, and, since the peak of interest (2) overlaps with peak 1 containing a strange adduct, it would be hard to purify. Da. The reduction and free cysteine essay, leads to removal of the adduct, but the resulting peak is the alkylated product, meaning the ring is not formed. Peak 2 has the correct mass, and the free cysteine essay causes almost no shift, meaning that the ring is formed in the majority of the product. Peak 2 is also easily separated from other products by HPLC, making it an attractive candidate future work.

g) HPLC spectrum
No major products could be purified for construct g, which is an indication that no dehydration can occur in this construct. Lack of dehydration and or ring formation leads to degradation during the long expression time for these constructs.

Control free cysteine assay unmodified construct a
Because the results of the free cysteine essay of construct a didn't completely agree with the mass shift of the TCEP reduction (i.e., the fast majority rather than all of the peptide shifted), a free cysteine essay was done on construct a expressed without MrsM, that should thus always result in a shift resembling the alkylation of two cysteines. As can be seen in the top figure, the fast majority of the product has been alkylated twice (7597.59 Da, 7.598,45 Da theoretical), with trace amounts of once alkylated product (7539.56 Da, 7.541,38 Da theoretical). Small amounts of product with a higher retention time can be seen in the lower figure. Here the amount of once alkylated product is higher, and trace amounts of non-alkylated product are observed (7483.54 Da, 7484,305 Da theoretical). The control points out that trace amounts of product are not fully alkylated, which should be considered a baseline of the free cysteine assays. And thus, from these experiments complete lack of ring formation cannot be confirmed. However, they are more than sufficient to compare ring formation efficiency between different mutants and confirm complete ring formation.
The same phenomenon can be seen in the control free cysteine essay of unmodified construct c (below). The cysteine residues in the majority of the product is completely alkylated (

S8. Activity tests all mutants
For all mutants, 8 µL of freeze-dried C-18 purified peptide, dissolved in 150 µL Milli-Q water, was digested by adding 1 µL of AprE-His, and setting the final volume to 10 µL by adding 1 µL of Milli-Q water, according to the previously described protocol 2 .
For the wild type control, containing MrsMD modified His6-MrsA, 2 µL of freeze-dried C-18 purified peptide, dissolved in 150 µL Milli-Q water was digested by adding 1 µL of AprE-His. For the control, the final volume was set to 10 µL by adding 7 µL of Milli-Q water. A nisin solution of 25 ng/µL was used as a positive control. Of all samples and controls, 9 µL was spotted on the activity plates.